Neuronal activity stimulates the expression of activity-dependent genes. This expression leads to molecular and structural changes within the neuron, which affect its connections with other neurons and underlie aspects of circuit development, learning and memory. Dysregulation of this response is thought to contribute to cognitive and neuropsychiatric disorders, including autism and schizophrenia. Recent studies indicate that non-coding enhancer sequences play a previously unappreciated role in regulating the activity- dependent transcriptional response. While enhancers are known to be important for the appropriate expression of genes in many tissues, little is known about their biological function in neurons. Previous studies of enhancer function or of activity-dependent gene expression have been hindered by the tedious genetic manipulations that are required to directly modify genetic sequences. The experiments put forward in this proposal aim to employ newly developed molecular tools to efficiently and reversibly test the temporal requirement for activity-dependent gene expression in neuronal connectivity, without gene targeting. Tools will be developed employing Transcription Activator-Like Effector (TALE) technology, which allows DNA- binding proteins to be easily designed to recognize a particular DNA sequence with high specificity. TALE protein fusions will be constructed for targeted and reversible regulatory sequence repression in primary mouse neurons. Once optimized, TALE protein fusions will be used to directly test the requirement for activity- dependent expression of Brain-derived neurotrophic factor (Bdnf) in maintaining normal inhibitory synapse numbers on mature neurons. These TALE-mediated expression switches will be widely useful to understand the roles of other regulatory elements, including enhancers, in brain development and function. In parallel, this proposal aims to begin investigating the function of novel activity-dependent enhancers in regulating neuronal adaptive responses. One such enhancer is located within the Bdnf gene, and is hypothesized to play a key role in the regulation activity-dependent Bdnf expression. Functional dissection of this novel enhancer sequence will yield mechanistic insight into its activity-dependent nature, while Bdnf gene variants harboring mutant enhancer sequences will be introduced into cortical neurons by BAC transfection in order to test this enhancer's role in the regulation of Bdnf expression profiles. These two lines of study will initiate and enable a more comprehensive analysis of activity-dependent enhancers as regulators of brain development and neuronal plasticity.